Forkhead Box (Fox) transcription factors Foxml and Foxfl are expressed in developing pulmonary endothelial cells and play an essential role in lung development. Deficiency of either one of these Fox genes is associated with severe defects in formation of peripheral pulmonary capillaries as well as abnormalities in many organs causing embryonic lethality. Our hypothesis is that Foxml and Foxfl regulate transcription of genes critical for proliferation and differentiation of endothelial cells during pulmonary vascular development. In order to test this hypothesis, we will use the Tie2 promoter driven Tamoxifen-inducible Cre recombinase (Tie2-Cre-ERT2) transgene to mediate developmental-stage-specific deletion of either Foxfl or Foxml LoxP/LoxP (fl/fl) targeted allele in endothelial cells. In Aim 1, we describe plans to use Tie2-Cre-ERT2 Foxml fl/fl embryonic lungs and cultured pulmonary endothelial cells derived from these embryos to determine whether Foxml is required for endothelial cell proliferation and differentiation during lung vascular development. In Aim 2, we will develop a mouse containing a Foxfl fl/fl targeted allele and use the Tie2-Cre- ERT2 transgene to mediate temporal endothelial-specific deletion of the Foxfl fl/fl at different stages of lung development. We will use Tie2-Cre-ERT2 Foxfl fl/fl mice to determine whether Foxfl directly regulates endothelial cell differentiation and identify a time period during which Foxfl function is required to elicit proper vascular lung development and to regulate Foxfl target genes. We have shown that Foxfl regulates expression of genes involved in Notch-2 signaling and that the Notch-2 pathway is critical for normal development of lung microvasculature. In Aim 3, we propose to generate mice with the endothelial-specific Tie-2 promoter driven Notch-2 transgene. These Tie-2 Notch-2 mice will be bred with Foxfl mice to determine whether increased Notch-2 receptor levels in embryonic Foxfl lung will restore normalcy to the development of alveolar microvasculature and prevent perinatal pulmonary hemorrhage in Foxfl newborn mice. Completion of the proposed studies will enable us to determine the role of Foxfl and Foxml proteins in endothelial cells during lung development. These Fox proteins may also provide novel targets in genetic screening, diagnosis and treatment of human pediatric lung diseases including perinatal pulmonary hemorrhage and respiratory distress syndrome of premature born babies.